The invention relates to the field of diagnostic assays and assays for identifying patients at risk for development of glaucoma.
The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
Glaucoma is the second most common cause of blindness in the United States. It is estimated that some two million Americans have glaucoma, with half of those suffering unaware of the presence of the disease. Primary open-angle glaucoma (xe2x80x9cPOAGxe2x80x9d) is the most common form of glaucoma, accounting for some 60 to 70% of all glaucomas.
POAG is characterized by obstruction of the normal aqueous outflow of fluids through the trabecular meshwork, canal of Schlemm, intrascleral channels, and episcleral and conjunctival veins. In open-angle glaucoma, this obstruction exists despite an angle that appears open. Generally, a patient that has not been otherwise diagnosed as having glaucoma first becomes aware of the disease due to losses in the visual field. By this point, the degree of optic nerve atrophy resulting from the disease may be quite severe, and is irreversible. Thus, early diagnosis and treatment play a key role in patient management.
Several risk factors have been identified as being related to POAG, including elevated intraocular pressure (xe2x80x9cIOP,xe2x80x9d 50% of patients present with an IOP of  less than 22 mm Hg), increased age (POAG is 6xc3x97 more common in persons  greater than 60 years of age), a family history of the disease (a 15xc3x97 increased chance of developing glaucoma), race (African Americans are at an increased risk for more serious disease), diabetes, hypertension, myopia, and the use of corticosteroids.
While elevated IOP is a primary risk factor for development of POAG, about ⅙ of patients exhibit an IOP within the normal range. Additionally, there is currently no reliable method for predicting which patients presenting with elevated IOP will progress to POAG. Recently, the relationship of the trabecular meshwork-inducible glucocorticoid response (xe2x80x9cTIGRxe2x80x9d) gene and protein have been studied for possible associations with glaucoma and related diseases. See, e.g., U.S. Pat. Nos. 5,861,497, 5,916,778, 5,925,748, and 6,248,867; Morissette et al., Nat. Genet. 19: 319-21 (1998); Brezin et al., Am. J. Med. Genet. 76: 438-45 (1998); Shimizu et al., Am. J. Ophthalmol. 130: 165-77 (2000); Nguyen et al., J. Biol. Chem. 273: 6341-50 (1998); and Lindblad-Toh et al., Nat. Genet. 24: 381-6 (2000). Each publication and patent in the foregoing section is hereby incorporated by reference in its entirety, including all tables, figures, and claims.
The present invention is drawn to methods and compositions for the screening of samples for one or more TIGR polymorphisms. The sample can be a biological sample, such as a sample from a subject. The invention can be used to determine which of a plurality of TIGR polymorphisms are present in the genome of a subject. Preferably, a plurality of different samples are assayed, each in its own individual reaction mixture, and/or several different polymorphisms in the TIGR gene are assayed in that single reaction mixture. Thus, a plurality of samples may be simultaneously assayed for several different TIGR polymorphisms in a single cycle (batch run) of the assay.
In a first aspect, the invention provides methods of testing for the presence of one or more polymorphisms of a TIGR gene, in one or more samples comprising TIGR nucleic acids, by generating a labeled nucleic acid that provides a means of identifying a particular polymorphism, thus distinguishing that polymorphism from other polymorphisms that might be present in the same gene. The particular polymorphism may be identified, for example, by determining both the length of the labeled nucleic acid and the identity of a distinctively labeled nucleotide incorporated at an end of the nucleic acid.
In preferred embodiments, these methods comprise one or more of the following steps: (a) preparing a reaction mixture that contains (i) sample nucleic acid suspected of containing a TIGR nucleic acid sequence, (ii) a nucleic acid polymerase, (iii) one or more extension primers, wherein the extension primers comprise nucleotide sequences that terminate at positions located one nucleotide 3xe2x80x2 from the positions of one or more preselected polymorphism(s) of interest, and (iv) one or more labeled dideoxynulceotide triphosphates, or ddNTPs; (b) incubating the reaction mixture under conditions such that extension primers that hybridize to the TIGR nucleic acids are labeled by addition of one of the ddNTPs comprising a label to the 3xe2x80x2-end of the detection primer, in order to generate one or more labeled oligonucleotides; and (c) detecting a signal from the labeled oligonucleotides. The presence of a specific polymorphism can be identified by the presence of a distinctive signal at a position in the sequence of the extended nucleic acid.
In certain embodiments, TIGR nucleic acid obtained from a sample is amplified to provide an amount of TIGR nucleic acid sufficient for primer extension to determine the presence or absence of one or more polymorphic forms of TIGR in the original sample. While the exemplary methods described hereinafter relate to amplification using the polymerase chain reaction (xe2x80x9cPCRxe2x80x9d), numerous other methods are known in the art for amplification of nucleic acids (e.g., isothermal methods, rolling circle methods, etc.). The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods.
The phrase xe2x80x9cTIGR nucleic acidxe2x80x9d refers to any nucleic acid containing sequences directly associated with production of the TIGR protein, including TIGR genomic DNA, TIGR-encoding hnRNA, mature TIGR-encoding mRNA, or amplification products thereof. Any TIGR nucleic acid may be the subject of the methods described herein. In certain embodiments for example, a TIGR RNA may be reverse transcribed into DNA, and the DNA subjected to the analysis methods described hereinafter. In preferred embodiments, the methods are applied to TIGR gene sequences.
The phrases xe2x80x9cTIGR gene sequences,xe2x80x9d xe2x80x9cTIGR genomic DNA,xe2x80x9d and xe2x80x9cTIGR genexe2x80x9d as used herein refer to the nucleic acid unit present in the genome of an animal, preferably a human, encoding the TIGR protein, and includes both the TIGR coding sequence and the upstream enhancer and promoter regions operably associated with the TIGR coding sequence in the genome.
The term xe2x80x9cbiological samplexe2x80x9d as used herein refers to a sample obtained from a biological source, e.g., an organism, cell culture, tissue sample, etc. A biological sample can, by way of non-limiting example, consist of or comprise blood, sera, urine, feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample and/or chorionic villi. Convenient biological samples may be obtained by, for example, scraping cells from the surface of the buccal cavity.
The term xe2x80x9csubjectxe2x80x9d as used herein refers to any eukaryotic organism. Preferred subjects are fungi, invertebrates, insects, arachnids, fish, amphibians, reptiles, birds, marsupials and mammals. A mammal can be a cat, dog, cow, pig, horse, ox, elephant, simian. Most preferred subjects are humans. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term xe2x80x9canimalsxe2x80x9d includes prenatal forms of animals, such as fetuses.
As used herein, a xe2x80x9cplurality of samplesxe2x80x9d refers to at least two. Preferably, a plurality refers to a relatively large number of samples. A plurality of samples is from about 5 to about 500 samples, preferably about 25 to about 200 samples, most preferably from about 50 to about 200 samples. Samples that are processed in a single batch run of the method of the invention are usually prepared in plates having 24, 48, 96, 144, or 192 wells. The term xe2x80x9csamplesxe2x80x9d includes samples per se as well as controls, standards, etc. that are included in a batch run.
A xe2x80x9cpreselected TIGR polymorphismxe2x80x9d is a TIGR nucleic acid sequence that has been selected for testing by the methods of the invention. Examples of preselected TIGR polymorphisms include wild type TIGR, and single base polymorphisms referred to herein as MT-1 (a promoter mutation), and T377M, E423K, and N480K (all exon 3 mutations). The MT-1 mutation replaces C with G at position 906 in the promoter sequence of TIGR (FIG. 2). The T377M mutation replaces C with T at position 468 in the TIGR coding sequence, leading to a substitution of Thr with Met in the TIGR protein; the N480K mutation replaces C with A at position 778 in the TIGR coding sequence, leading to a substitution of Asn with Lys in the TIGR protein; and the K423E mutation replaces A with G at position 605 in the TIGR coding sequence, leading to a substitution of Lys with Glu in the TIGR protein (FIG. 1).
The assays described herein can be used to rapidly determine which of a selected group of polymorphic TIGR nucleic acid forms are present in a sample comprising TIGR nucleic acid. By xe2x80x9crapidxe2x80x9d it is meant that the length of time that is taken to carry out a single batch run of the assay, from the moment a reaction mixture comprising nucleic acid is prepared to the moment a signal can be detected, is from about 1 second to about 10, 15 or 30 seconds, about 1, 5, 10 or 30 minute(s), or 1, 3, 5, 8, 24 or 48 hour(s). When samples are from multiple subjects, the assays can be used to determine the TIGR genotype of each subject.
By xe2x80x9cdistinctively labeledxe2x80x9d, it is meant that each type of member of a set is labeled with a label that can be distinguished from the labels used for other members of the set. For example, in a set of distinctively labeled nucleotides (e.g., dideoxy NTPs, or ddNPTs), each type of xe2x80x9cNxe2x80x9d (nucleotide) is labeled with a label that can be distinguished from the other types of labels. Thus, for example, if four labels designated 1, 2, 3, and 4 are used to label the four types of ddNTPs, each ddATP molecule is labeled with label xe2x80x9c*1xe2x80x9d, each ddTTP molecule is labeled with label xe2x80x9c*2xe2x80x9d, each ddCTP molecule is labeled with label xe2x80x9c*3xe2x80x9d, and each ddGTP molecule is labeled with label xe2x80x9c*4xe2x80x9d. In some aspects of the invention, the distinctive label is a fluorescent label.
The skilled artisan will understand that, if one wishes to determine if a specific genotype is present in a sample, e.g., a xe2x80x9cTxe2x80x9d in a position in the TIGR sequence that would be a xe2x80x9cGxe2x80x9d in the wild-type sequence, one need only provide a single labeled ddNTP, in this case ddTTP, with an appropriate extension primer. If the xe2x80x9cTxe2x80x9d polymorphism is present, the labeled ddTTP will be incorporated into the 3xe2x80x2 end of the extension primer. In contrast, if the wild-type sequence is present, no labeled extension primer will be created. Depending on the polymorphisms selected for analysis, from one to four labeled ddNTPs may be required to perform an assay. One may also choose to include all four ddNTPs in a reaction for convenience, or so that even wild-type sequences become labeled.
As used herein, xe2x80x9cprimer extensionxe2x80x9d refers to the enzymatic extension of the three-prime (3xe2x80x2) hydroxy group of an extension primer, which is an oligonucleotide that is paired in a duplex to a template nucleic acid. For an example of primer extension as applied to the detection of polymorphisms, see Fahy et al., Mutliplex fluorescence-based primer extension method for quantative mutation analysis of mitrochondrial DNA and its diagnostic application for Alzheimer""s disease, Nucleic Acid Research 25:3102-3109, 1997. The extension reaction is catalyzed by a DNA polymerase. By xe2x80x9cDNA Polymerasexe2x80x9d it is meant a DNA polymerase, or a fragment thereof, that is capable of catalyzing the addition of bases to a primer sequence in a sequence-specific fashion. A DNA polymerase can be an intact DNA polymerase, a mutant DNA polymerase, an active fragment from a DNA polyerase, such as the Klenow fragment of E. coli DNA polymerase, and a DNA polymerase from any species, including but not limited to thermophilic organisms.
Extension of the 3xe2x80x2 end of the oligonucleotide generates an oligonucleotide having a length greater than the extension primer and having a sequence that is the reverse complement of the template nucleic acid. If one of the nucleotides in the added sequence is labeled, then the extended oligonucleotide becomes labeled.
Preferably, an extension primer has a nucleotide sequence that binds in a complementary fashion to a portion of a nucleic acid sequence that encodes or modulates the expression of the TIGR gene, or to the complement of such a sequence. Extension primers must be of a length sufficient to provide specific binding to the target sequence of interest. Such primers comprise an exact complement to the sequence of interest for 15 to 75 nucleotides in length, preferably 17 to 50 nucleotides in length, and more preferably from 20 to 30 nucleotides in length. The extension primer sequence has a 3xe2x80x2 terminus that pairs with a nucleotide base that is, in the sample nucleic acid to which the primer is hybridized, 5xe2x80x2 from the site of one or more bases in the sequence of interest that represent a polymorphism in a gene. Suitable extension primers are described herein, and may be one of the sequences set forth in SEQ ID NOS:1-4.
In addition to the sequence that ensures hybridization to the target site, an extension primer may have additional nucleotides added to the 5xe2x80x2 end that need not participate in specific binding. Thus, such primers may extend for 15 to 75 nucleotides in length, preferably 17 to 50 nucleotides in length, and more preferably from 20 to 30 nucleotides in length, only a subset of which is an exact complement to the sequence of interest. In these embodiments, the exact complement may extend for at least 15 nucleotides, more preferably for at least 17 nucleotides, and most preferably for at least 20 nucleotides to ensure specific hybridization of the extension primer. Thus, an extension primer may contain one of the sequences set forth in SEQ ID NOS:1-4 at the 3xe2x80x2 end, with additional nucleotides at the 5xe2x80x2 end which may or may not be complementary to the TIGR sequence of interest.
In the following diagram of a primer extension reaction, four different ddNTPs, each distinctively labeled, are present in the reaction mixture as designated by dd(A*1)TP, dd(T*2)TP, dd(C*3)TP and dd(G*4)TP, where *1, *2, *3 and *4 represent different labels. In the diagram, the polymorphism in the nucleic acid being tested is indicated by an underlined nucleotide, and the extension primer sequence is italicized. Only one ddNTP, ddTTP, can be added to the 3xe2x80x2 end of the extension primer, because thymine (T) is the only base that pairs with adenosine (A). The addition of the dd(T*2)TP to the 3xe2x80x2 of the primer prevents any further primer extension because it is a dideoxy, chain-terminating ddNTP. Thus, the only primer that is 3xe2x80x2 extended is labeled with label *2. Detection of the signal from label *2 indicates that the A polymorphism is present in the sample:
As discussed herein, an amount of nucleic acid sufficient for primer extension can, but need not be, prepared by amplification, e.g., via PCR using amplification primers. As a non-limiting example, appropriate amplification primers include, but are not limited to, those having sequences set forth in SEQ ID NOS:5-9.
For each reaction mixture, the amount of the nucleic acid sufficient for primer extension can be determined by obtaining a sample comprising nucleic acid and determining the concentration of nucleic acid therein. One skilled in the art will be able to prepare such samples to a concentration and purity necessary to practice the invention, and to estimate the amount of a specific sample that should be added to a particular reaction mixture. A failure to detect a signal in the method of the invention may signify that, among other things, an inadequate amount of nucleic acid has been added to a reaction mixture. Those skilled in the art will be able to trouble-shoot failed batch runs and adjust the contents of the reaction mixtures and/or conditions of the run accordingly. Control samples, both positive and negative, can be included in the batch runs to confirm that appropriate amounts of nucleic acid are present.
One or more of steps of the assays described herein, in any combination, are preferably performed in an automated fashion, typically using robotics, in order to provide for the processing of a large number of samples in a single batch run. Preferred forms of automation will provide for the preparation and separation of a plurality of labeled nucleic acids in small volumes. The term xe2x80x9csmall volumesxe2x80x9d refers to volumes of liquids less than 2 ml, e.g., any volume from about 0.001 picoliters or about 0.001 xcexcl, to any volume about 2 ml, 500 xcexcl, 200 xcexcl, 100 xcexcl, 10 xcexcl, 1 xcexcl, 0.1 xcexcl, 0.01 xcexcl or 0.001 xcexcl.
The set of distinctively labeled oligonucleotides generated by the methods described herein can be separated from each other so that each is mobilized in a manner that relates to each of their specific positions in the respective nucleotide sequence, and the detection of the distinctive signals generated from the distinctively labeled oligonucleotides occurs during or after the mobilization. Members of the set of distinctively labeled oligonucleotides can be separated from each other so that each is mobilized by electrophoresis. A preferred form of electrophoresis is capillary electrophoresis, but any form of electrophoresis that allows for the separation of a plurality of labeled nucleic acids in small volumes by automated or semi-automated methods and devices may be used.
In additional aspects of the present invention, polymorphic forms of TIGR assayed according to the invention can be used to diagnose subjects suffering from glaucoma or to identify subjects that are at increased risk for developing glaucoma. In preferred embodiments, such subjects may also exhibit one or more additional risk factors for glaucoma, including elevated IOP (xe2x89xa722 mm Hg, more preferably 22-30 mm Hg, most preferably 27-30 mm Hg), increased age (xe2x89xa760 years of age), a family history of glaucoma, diabetes, hypertension, myopia, and the use of corticosteroids. In certain embodiments, such subjects exhibit a normal IOP (11-21 mm Hg).
In other aspects, the results from the assays of the invention can be used to initiate or design a regimen of treatment, based on an increased risk for developing glaucoma. Such treatment may include one or more of the following: surgical or laser treatments, installation of shunts, treatment with miotics (such as pilocarpine, carbachol, physostigmine, demecarium, and isofluorophate), treatment with carbonic anhydrase inhibitors (such as acetazolamide and methazolamide), adrenergic agonists (such as epinephrine, dipivefrine, and xcex12-specific agonists such as apraclonidine), xcex2-blockers (such as betaxolol and metipranolol), prostaglandin analogs, and osmotic diuretics.
In yet other aspects, the present invention relates to one or more oligonucleotide molecules that are amplification primers and/or extension primers for use in the present invention. Preferably, the extension primers comprise sequences selected from the group consisting of SEQ ID NOS: 1-4, and the amplification primers comprise sequences selected from the group consisting of SEQ ID NOS: 5-8. Preferably, the oligonucleotide molecules of the present invention are purified, and most preferably substantially pure molecules.
As used herein, the term xe2x80x9cpurifiedxe2x80x9d in reference to oligonucleotides does not require absolute purity. Instead, it represents an indication that the sequence is relatively more pure than in the natural environment. Such oligonucleotides may be obtained by a number of methods including, for example, laboratory synthesis, restriction enzyme digestion or PCR. A xe2x80x9cpurifiedxe2x80x9d oligonucleotide is preferably at least 10% pure. A xe2x80x9csubstantially purifiedxe2x80x9d oligonucleotide is preferably at least 50% pure, more preferably at least 75% pure, and most preferably at least 95% pure.
In further aspects, the present invention also relates to kits for performing the methods described herein. Preferably, such kits contain one or more extension primers in an amount sufficient to perform at least one assay for determining the presence or absence of a particular polymorphic form of TIGR in a sample. More preferably, such kits contain extension primers in an amount sufficient to perform at least one assay for determining the presence or absence of at least two, and most preferably at least four, different polymorphic forms of TIGR in a sample. Preferably, the extension primers comprise sequences selected from the group consisting of SEQ ID NOS: 1-4. In certain embodiments, the kits also contain amplification primers in an amount sufficient to perform a PCR amplification of the polymorphism region(s) of interest in the assay. Preferably, the amplification primers have sequences selected from the group consisting of SEQ ID NOS: 5-8. In certain other embodiments, the kits may also contain an instruction manual providing instructions for use of the extension probes, and amplification probes if present in the kit.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.